The long-term objectives of this research are to understand the mechanisms that regulate hair cell regeneration in the avian inner ear and to apply this knowledge to induce regeneration in the mammalian cochlea. Studies from the previous three funding periods of this grant have shown that the auditory epithelium in the postembryonic avian cochlea is mitotically quiescent. However, in response to sound exposure or aminoglycoside administration, the nonsensory supporting cells emerge from quiescence and either transdifferentiate directly into hair cells or divide to produce new hair cells and supporting cells that contribute to the structural and functional recovery of the cochlea. In this proposal, we will examine the mechanisms by which the proliferative state of supporting cells in the avian cochlea is activated by the dying hair cells. Our hypothesis is that apoptosis in hair cells releases signals that induce the supporting cells to directly transdifferentiate into hair cells or to exit quiescence and proliferate. We propose that this choice is determined by the number of hair cells lost in the vicinity of the supporting cells. If only a few hair cells are lost, then direct transdifferentiation is activated. If a majority of the hair cells in the region are lost, then mitotic proliferation is induced. The specific aims of this proposal will examine: 1) whether inhibition of cell death pathways can lead to hair cell rescue in cochlear organ culture preparations and how this affects the regenerative responses of the supporting cells; 2) the mechanisms that the supporting cells use to choose between direct transdifferentiation and mitotic proliferation and how this is impacted by the extent of hair cell loss; and 3) the role that the unconventional myosins VI and Vila play in the disassembly and ejection of dying hair cells from the sensory epithelium. The studies addressed by these specific aims will enable us to manipulate the hair cell death pathways and determine whether this can rescue the sensory cells. They will also inform us of how the dying hair cells regulate the regenerative pathways that the supporting cells undertake to restore the sensory epithelium. Ultimately, our goal is to identify similar mechanistic responses in mammalian hair cells and supporting cells, and determine whether biochemical interventions can be used to encourage mammalian hair cells to follow similar regenerative pathways after cochlear damage.